Neponin detection test strip and preparation method and application thereof

Abstract

The invention discloses a continuous protein rapid detection test strip and a preparation method thereof, the adopted technology is an immunochromatography method, and the reaction principle is a competition method. The test strip comprises a bottom plate, and a blood filter pad, a conjugate pad 1, a conjugate pad 2, a nitrocellulose membrane and a water absorption pad which are arranged on the bottom plate, the blood filtering pad is prepared after being treated by the pretreatment liquid; the conjugate pad 1 is coated with a zonulin polyclonal antibody marked by fluorescent microspheres and a chicken IgY antibody marked by fluorescence; the conjugate pad (2) is coated with a biotinylated zonulin antigen; the nitrocellulose membrane is provided with a detection line and a quality control line, and the detection line is coated with an anti-biotin antibody; and the quality control line is coated with a rabbit anti-chicken IgY antibody. According to the invention, not only can whole blood, serum and plasma samples be directly and rapidly detected, but also an excrement sample can be detected, the structure is simple, small and portable, and a simpler, more convenient and faster method is provided for rapid detection and screening of zonulin.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a zonulin test strip, its preparation method, and its application. Background Technology

[0002] Linalool is a human protein analog derived from the banded occlusive toxin of Vibrio cholerae, used to regulate tight junctions in the digestive tract.

[0003] Tight junctions bind to specific receptors on the surface of the intestinal epithelium and trigger a series of biochemical events, resulting in the disintegration of tight junctions and increased permeability of the intestinal epithelium, allowing certain substances to pass through and activating an immune response.

[0004] Currently, the most common method for detecting zonulin is enzyme-linked immunosorbent assay (ELISA). However, ELISA is cumbersome, involves many sample pretreatment steps, requires a long detection time, and the reagents need to be stored at 2–8°C, which does not meet the requirements for rapid point-of-care testing.

[0005] This invention provides an immunochromatographic test strip for the quantitative detection of zonulin, which is simple to operate, has a short reaction time, and does not require special sample treatment. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a convenient zonulin test strip.

[0007] A zonulin test strip includes a base plate and, sequentially attached to the base plate along its length, a blood filter pad, a conjugate pad 1, a conjugate pad 2, a coating membrane, and an absorbent pad; the blood filter pad is prepared by pretreatment solution 1; the conjugate pad 1 is coated with fluorescent microspheres labeled with zonulin polyclonal antibody and chicken IgY antibody labeled with fluorescent microspheres; the conjugate pad 2 is coated with biotinylated zonulin antigen; the coating membrane has a detection line and a control line sequentially arranged, the detection line is coated with anti-biotin antibody, and the control line is coated with rabbit anti-chicken IgY; the pretreatment solution 1 includes 0.5% to 1.5% surfactant; the fluorescent microspheres are lanthanide time-resolved microspheres.

[0008] The purpose of this invention is to provide a rapid detection kit for zonulin, which includes the zonulin detection test strip described above. Attached Figure Description

[0009] Figure 1 This is a schematic diagram of the structure of the zonulin test strip in Example 1.

[0010] Figure 2 This is the calibration curve established by the ratio of detected values ​​to concentration in Example 2.

[0011] Figure 3 The correlation between the test results of the reagent card of the present invention and the detection results of the enzyme immunoassay kit when testing whole blood samples in Example 2 is shown.

[0012] Figure 4 The correlation between the test results of the reagent card of the present invention and the detection results of the enzyme immunoassay kit when detecting fecal samples in Example 2 is shown. Detailed Implementation

[0013] Experimental methods in the following embodiments of the present invention, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0014] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0015] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0016] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0017] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0018] The present invention will be further described in detail below with reference to specific embodiments.

[0019] Some embodiments of the present invention provide a zonulin test strip, comprising a base plate and, sequentially attached along the length of the base plate, a blood filter pad, a conjugate pad 1, a conjugate pad 2, a coating membrane, and an absorbent pad; the blood filter pad is prepared after treatment with a pretreatment solution 1; the conjugate pad 1 is coated with fluorescent microspheres labeled with zonulin polyclonal antibody and fluorescent microspheres labeled with chicken IgY antibody; the conjugate pad 2 is coated with biotinylated zonulin antigen; the coating membrane is sequentially provided with a detection line and a control line, the detection line is coated with anti-biotin antibody, and the control line is coated with rabbit anti-chicken IgY; the pretreatment solution 1 includes 0.5% to 1.5% surfactant; the fluorescent microspheres are lanthanide time-resolved microspheres.

[0020] In some embodiments, the pretreatment solution 1 is a 0.5M to 1.5M Tris buffer solution at pH 7.4 containing 0.5% to 1.5% surfactant.

[0021] In some embodiments, the pretreatment solution 1 is a 0.9M to 1.2M Tris buffer solution at pH 7.4 containing 0.8% to 1.2% surfactant.

[0022] In some embodiments, the pretreatment solution 1 described above is a 1M Tris buffer at pH 7.4 containing 1% surfactant.

[0023] In some embodiments, the surfactant in the pretreatment solution 1 is selected from Triton X-100, Pluracare 1307, Tween-80, and Tween 20. Pluracare 1307, also known as surfactant S9 or Tetronic 1307, is a propylene oxide-ethylene oxide-vinyl diamine copolymer and is an amphoteric surfactant. The surfactant functions to reduce or eliminate the influence of the sample matrix or non-specific reactions, and to increase the flow and washing effect of the liquid.

[0024] In some of the embodiments, the fluorescent microspheres labeled with the zonulin polyclonal antibody described above are time-resolved fluorescent microspheres with a particle size of 160-400 nm that have been modified with carboxyl groups.

[0025] In some embodiments, the mass ratio of the carboxyl-modified time-resolved fluorescent microspheres to the zonulin polyclonal antibody labeled with the above-mentioned zonulin polyclonal antibody is 1:0.02 to 1:0.1.

[0026] In some embodiments, the zonulin polyclonal antibody-labeled fluorescent microspheres are diluted to a concentration of 0.1 mg / ml to 1 mg / ml using a labeling antibody diluent, and sprayed onto the conjugate pad at a rate of 3 μl to 5 μl / 1 cm × 3 cm. The labeling antibody diluent is 0.05 M TBS with a pH of 7.2.

[0027] In some embodiments, the concentration of the above-mentioned chicken IgY antibody-labeled fluorescent microspheres is 0.01 mg / mL-0.05 mg / mL, and they are sprayed onto the conjugate pad at a rate of 3 μl-5 μl / 1 cm × 3 cm.

[0028] In some embodiments, the concentration of the anti-biotin antibody coated on the detection line is 0.5 mg / ml to 2 mg / ml, and it binds to the coated membrane at a rate of 0.5 μl to 1.5 μl / 1 cm.

[0029] In some embodiments, the concentration of rabbit anti-chicken IgY antibody coated on the above-mentioned quality control line is 0.2 mg / ml to 1 mg / ml, and it binds to the coated membrane at a rate of 0.5 μl to 1.5 μl / 1 cm.

[0030] Some embodiments of the present invention also provide a zonulin detection kit, which includes the above-described zonulin detection test strip.

[0031] In some embodiments, the above-described zonulin detection kit is used to detect the content of zonulin in human fecal or serum, plasma, or whole blood samples.

[0032] Example 1: Preparation and Detection of Test Strips

[0033] I. Preparation of test strips

[0034] (I) Preparation of bonding pad 1

[0035] (1) Take 100 μL of 10% carboxyl-modified fluorescent microspheres, wash with MES buffer (0.1 M, pH 5.0) and centrifuge. Discard the supernatant, resuspend with 1 mL MES buffer (0.1 M, pH 5.0), add EDC to a final concentration of 10 mM, add NHS to a final concentration of 10 mM, and activate at room temperature in the dark for 30 minutes.

[0036] (2) Wash the activated microspheres with 100mM MES buffer (0.1M, pH 6.2), add 50μL of 1mg / mL zonulin polyclonal antibody (purchased from Youkang Biotechnology), react at room temperature in the dark for 30 minutes, then centrifuge to separate, remove the non-bound antibody, and resuspend.

[0037] (3) Add BSA to a final concentration of 1% for blocking. After completion, wash and resuspend with 0.05M TBS buffer at pH 7.2 to obtain zonulin antibody-labeled fluorescent microspheres, and store at 4°C for later use.

[0038] (4) The microsphere complex prepared in (3) above and the chicken IgY antibody labeled with fluorescent microspheres (purchased from Youkang Biotechnology) were diluted 20 times with the labeling antibody diluent (0.05M TBS, pH7.2). The concentrations of the fluorescent microspheres labeled with zonulin antibody and the fluorescent microspheres labeled with chicken IgY antibody were 0.3 mg / ml and 0.03 mg / ml, respectively. They were sprayed onto a glass fiber membrane at a spraying amount of 4 μL / cm and dried at 37°C overnight to obtain conjugate pad 1.

[0039] (II) Preparation of bonding pad 2

[0040] (1) Take 50 μL of 1 mg / ml biotin dissolved in DMF and add it to 0.05 M TBS buffer at pH 7.2 containing 0.3 mg zonulin antigen (purchased from Youkang Biotechnology). React at room temperature in the dark for 1 hour, dialyze overnight at 4°C, and store for later use.

[0041] (2) The complex prepared in (1) above was sprayed onto a glass fiber membrane at a concentration of 0.08 mg / mL, with a spraying amount of 4 μL / cm. The membrane was dried at 37°C overnight to obtain the conjugate pad 2.

[0042] (III) Preparation of blood filtering pads

[0043] The glass fiber membrane was treated with pretreatment solution 1 containing 1% Tween-20 and 0.1M Tris (pH 7.4) and dried overnight in a drying oven at 37°C to obtain the sample pad.

[0044] (iv) Preparation of test strips

[0045] 1. Preparation of nitrocellulose membrane (NC membrane) 4 coated with detection line (T line) and control line (C line):

[0046] Antibiotin antibody (T-line antibody) (purchased from Yocon Biotechnology) was diluted to 1 mg / ml and rabbit anti-chicken IgY antibody (C-line antibody) was diluted to 0.8 mg / ml respectively with coating protein dilution buffer (50 mM PBS, pH 7.4). The spraying volume was set to 1 μL / cm under the condition of 50% to 65% humidity. After streaking, the antibody was dried at 50°C for 2 hours and then stored in sealed bags under the condition of less than 35% humidity for later use.

[0047] 2. Assembly of the test strip card: On a PVC board, sequentially attach the treated sample pad, conjugate pad 1, conjugate pad 2, nitrocellulose membrane coated with detection lines and control lines, and absorbent pad. After assembly, a large test strip board is obtained. Cut it into 3-5mm wide pieces as required, and insert the test strips into the plastic card to form the test strip card. See the structural diagram below. Figure 1 .

[0048] II. Testing

[0049] 1. Operating Steps

[0050] Before testing, equilibrate all reagents to room temperature (15–30°C). The specific steps are as follows:

[0051] 1) Turn on the fluorescence immunoassay analyzer;

[0052] 2) Insert the ID card;

[0053] 3) Add the sample to the sample diluent (0.05M TBS, pH 7.2) and mix thoroughly;

[0054] 4) Prepare the reagent card, add the mixed liquid droplet into the sample well of the reagent card, and be careful not to inhale air bubbles when taking the sample. Let it stand at room temperature for 15 minutes.

[0055] 5) Follow the instructions in the instrument manual, insert the test card into the analyzer, and read the test results after 15 minutes.

[0056] Example 2 Sample Detection

[0057] The principle of this invention is based on a competitive method of fluorescence immunochromatography. The antigen in the sample mixes with zonulin polyclonal antibody-labeled fluorescent microspheres in conjugate pad 1, diffuses along conjugate pad 2, and competes with the biotinylated zonulin antigen coated on conjugate pad 2 for the zonulin antibody-labeled microspheres on conjugate pad 1, forming an antigen-antibody-fluorescent microsphere complex. This complex continues to diffuse along the nitrocellulose membrane, and upon reaching the detection zone, is captured by anti-biotinylated antibody immobilized on the T-line, forming the T-line. Chicken IgY in the reaction system is captured by rabbit anti-chicken IgY antibody immobilized on the C-line, forming the control line. Ultimately, the more antigen in the sample, the fewer the complex on the T-line, and the weaker the fluorescence signal. By measuring and analyzing the fluorescence signal of this complex using a matching instrument, the content of zonulin in the sample can be quantitatively determined.

[0058] (I) Calibration Curve

[0059] Using ligin antigen (purchased from UCON Biotechnology) as the test sample, the antigen was prepared into a series of concentrations (0.5, 1.0, 5.0, 10.0, 40.0, 80.0, 160 ng / mL) using sample diluent. 80-100 μL of each concentration (selected according to the width of the test strip) was added to the sample application area of ​​the test strip prepared in Example 1. After reacting at room temperature for 15 minutes, fluorescence detection was performed. The calibration curve established based on the ratio of detection values ​​to concentration is as follows: Figure 2 As shown, it exhibits good correlation, with a detection range of 0.5-160 ng / mL.

[0060] (II) Whole blood sample testing

[0061] Thirty-six clinical whole blood samples were collected and tested according to the method described in this invention, and compared with an enzyme immunoassay kit (purchased from Adico GmbH, Germany). The enzyme immunoassay kit uses serum separated from whole blood samples for simultaneous detection. The comparison results are as follows. Figure 3 As shown, the correlation coefficient R2 between the present invention and the clinical test results is 0.9707, indicating that the present invention has a good correlation with ELISA and high accuracy.

[0062] (III) Fecal Sample Testing

[0063] Forty-six clinical stool samples were collected and tested according to the method described in this invention. The results were compared with an enzyme immunoassay kit (purchased from Adico GmbH, Germany). The results are as follows: Figure 4 As shown in the figure, the correlation coefficient R² between the present invention and the ELISA results is 0.9772, indicating that the present invention has a good correlation with ELISA and high accuracy.

[0064] As can be seen from the above sample test results, the test strip of the present invention is applicable to both whole blood and stool samples.

[0065] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A zonulin test strip, characterized in that, The test strip uses immunochromatography, and its reaction principle is a competitive method. The test strip includes a base plate and, sequentially attached to the base plate along its length, a blood filter pad, a conjugate pad 1, a conjugate pad 2, a coating membrane, and an absorbent pad. The blood filter pad is prepared after treatment with pretreatment solution 1. The conjugate pad 1 is coated with fluorescent microspheres labeled with zonulin polyclonal antibody and fluorescent microspheres labeled with chicken IgY antibody. The conjugate pad 2 is coated with biotinylated zonulin antigen. The coating membrane has a detection line and a control line sequentially arranged on it. The detection line is located on the coating membrane near the conjugate pad 2 and is coated with anti-biotin antibody. The control line is located on the coating membrane near the absorbent pad and is coated with rabbit anti-chicken IgY.

2. The zonulin test strip according to claim 1, characterized in that... The pretreatment solution 1 is a 0.1M to 1.5M Tris buffer solution containing 0.5% to 1.5% surfactant, with a pH of 7.

4.

3. The zonulin test strip according to claim 2, characterized in that... The pretreatment solution 1 is a 0.1M Tris buffer containing 1.0% Tween 20 with a pH of 7.

4.

4. The zonulin test strip according to claim 1, characterized in that, The zonulin polyclonal antibody-labeled fluorescent microspheres have a coating concentration of 0.1 mg / ml to 1 mg / ml and a spraying volume of 4 μL / cm; the chicken IgY antibody-labeled fluorescent microspheres have a coating concentration of 0.01 mg / mL to 0.05 mg / mL and a spraying volume of 4 μL / cm.

5. The zonulin test strip according to claim 1, characterized in that, The biotinylated zonulin antigen was coated at a concentration of 0.02 mg / mL to 0.12 mg / mL, with a spraying volume of 4 μL / cm, and coated on the conjugate pad 2.

6. The zonulin test strip according to claim 1, characterized in that, The coating membrane is a nitrocellulose membrane, with a detection line and a control line sequentially arranged on the membrane. The concentration of antibiotin antibody coated on the detection line is 0.5 mg / ml to 2 mg / ml, and the spraying amount is 1 μL / cm. The concentration of rabbit anti-chicken IgY coated on the control line is 0.2 mg / ml to 1 mg / ml, and the spraying amount is 1 μL / cm.

7. The zonulin test strip according to claim 1, characterized in that, The fluorescent microspheres are lanthanide time-resolved fluorescent microspheres with a particle size of 160–400 nm that have been modified with carboxyl groups.

8. The zonulin test strip according to claim 1, characterized in that, The test strip is used to quantitatively detect the content of zonulin in human whole blood and fecal samples.

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